Compromise of the pulmonary endothelial cell (EC) barrier is induced by mechanical stress which is associated with ventilator-induced lung injury (VILI), This process leads to increased vascular permeability, alveolar flooding, leukocyte infiltration, hypoxemia, and increased morbidity and mortality. Project #3 scientists have previously identified cytoskeletal mechanisms of EC barrier regulation by barrier-protective strategies (oxidized phosphocholine, sphingosine 1-phosphate, physiologic shear stress and cyclic stretch (CS) and barrier-disruptive mechanical and chemical stimuli (thrombin, pathologic CS). In addition, these PPG investigator have described the critical involvement of small GTPases Rac and Rho in remodeling of EC cytoskeleton and cell contacts essential for EC barrier regulation. Our published results and preliminary data strongly suggest that magnitude-dependent modulation of Rac and Rho activities by CS significantly impacts agonist-induced EC permeability changes. The Project #3 hypothesis is that pathologic CS and vascular endothelial growth factor (VEGF), known to be elevated during VILI, promote lung endothelial barrier dysfunction via synergistic effects on Rho pathway-mediated EC permeability. These processes are counterbalanced by Rac-dependent mechanisms induced by barrier-protective stimuli such as hepatocyte growth factor (HGF) and physiologic stretch. We speculate that focal adhesions may act as mechanosensors and modulate small GTPase activities via specific paxillin interactions with Rac and Rho protein regulators. Specific Aim #1 will study synergistic effects between pathologic CS and VEGF on activation of Rhomediated EC barrier dysfunction. Specific Aim #2 will define barrier-protective strategies in amelioration of VILI-associated EC barrier dysfunction via changes in a balance between the Rho and Rac activities. Specific Aim #3 will study novel mechanisms of Rac/Rho regulation by mechanochemical factors via interactions between wild type paxillin (or/and paxillin containing the Gly73Ser polymorphism) interactions with modulators of Rac and Rho activity (GIT2, betaPIX, PAK1 and p190RhoGAP). These studies will uncover novel molecular mechanisms involved in the pathogenesis and resolution of ventilator-induced lung injury.